Method and apparatus for in vivo surveillance of circulating biological components

ABSTRACT

The invention relates generally to in vivo collection of circulating molecules, tumor cells and other biological markers using a collecting probe. The probe is configured for placement within a living organism for an extended period of time to provide sufficient yield of biological marker for analysis.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority under 35 U.S.C. §119(e) to U.S.Provisional Application No. 60/531,928 filed on Dec. 22, 2003, thedisclosure of which is incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to devices and methods for collectingand/or detecting biological components in vivo over a period of time.The detection and/or analysis of the biological components collected bythe devices may be performed in vivo or ex vivo.

2. Description of the Related Art

Cancer is one of the leading causes of disease, being responsible for563,700 deaths in the United States each year (Jemal A et al., Cancerstatistics, 2004, CA Cancer J. Clin. 2004 January-February; 54(1):8-29). For example, breast cancer is the most common form of malignantdisease among women in Western countries and, in the United States, isthe most common cause of death among women between 40 and 55 years ofage (Forrest A P, Screening and breast cancer incidence, J Natl CancerInst. 1990 Oct. 3; 82(19): 1525-6.). The incidence of breast cancer isincreasing, especially in older women, but the cause of this increase isunknown. Malignant melanoma is another form of cancer whose incidence isincreasing at a frightening rate, at least six fold in the United Statessince 1945, and is the single most deadly of all skin diseases (Jemal etal., 2004).

One of the most devastating aspects of cancer is the propensity of cellsfrom malignant neoplasms to disseminate from their primary site todistant organs and develop into metastases. The early spread of viabletumor cells is considered a hallmark in cancer progression. Despiteadvances in surgical treatment of primary neoplasms and aggressivetherapies, most cancer patients die as a result of metastatic disease.Animal tests indicate that a substantial frequency of circulating cancercells from solid tumors establish successful metastatic colonies(Fidler, 1993). Studies have found that the detection of circulatingmetastatic tumor cells and circulating tumor DNA in the blood of cancerpatients correlates with cancer progression. (Hoon D S, et al.,Molecular markers in blood as surrogate prognostic indicators ofmelanoma recurrence, Cancer Res. 2000 Apr. 15; 60(8): 2253-7, and TabackB, et al., Circulating DNA microsatellites: molecular determinants ofresponse to biochemotherapy in patients with metastatic melanoma, J.Natl. Cancer Inst. 2004 Jan. 21; 96(2): 152-6, herein incorporated intheir entirety by reference).

Thus, the detection of occult cancer cells, DNA and tumor markers in thecirculation is important in assessing the level of tumor progression andmetastasis. Because subclinical metastasis can remain dormant for manyyears, traditional surveillance measures such as radiological monitoringwith CT scans or MRI and nodal biopsy may lack the sensitivity to detectearly disease.

Notwithstanding the foregoing, there remains a need for improved methodsand devices for detecting biological components of disease.

SUMMARY OF THE INVENTION

In one embodiment of the invention, a biological surveillance probe fordetecting disease is provided. The probe comprises an elongate bodyhaving a proximal end and a distal end, a binding surface attached tothe elongate body, wherein the binding surface has a microconfigurationfor an increased surface area, and at least one binding partner attachedto the binding surface to bind at least one complementary target. Insome embodiments, the binding surface is a microporous surface or has atleast one laser-drilled hole. In some embodiments, the binding surfacebeen configured by vapor deposition, physical vapor deposition, chemicalvapor deposition, sputtering, reactive sputtering, sintering or vacuumdeposition. The binding surface may comprise a material selected from agroup comprising a microporous polymer, nanotube, metal, non-metal,ceramic or combination thereof. The elongate body may be a catheter bodyor a stent support. The binding surface may be a polymeric jacket. Insome embodiments, the probe may further comprise at least one opticallysensitive dye engaged to the binding surface, a fibrin-depositionresistant component, at least one anti-thrombotic agent or antimicrobialagent engaged to the binding surface. An atraumatic tip may be attachedto the distal end of the elongate body.

In another embodiment of the invention, a method for collectingbiological markers is provided. The method comprises the steps ofproviding a collecting probe comprising a microconfigured bindingsurface and at least one binding agent affixed to the binding surfacefor binding a marker, positioning at least a portion of the probe in ananatomical structure of a living organism; maintaining the probe in ageneral position for a specified period of time; and removing the probefrom the living organism. The method may further comprise the steps ofbinding at least marker at a first point in time; and binding at leastone marker at a second point in time. The method may also comprisebinding at least one marker at a about first peak in markerconcentration and binding at least one marker at about a second peak inmarker concentration. The method may further comprise analyzing theprobe for markers bound to the binding agent. The analyzing step may beperformed ex vivo.

Several embodiments of the present invention provides these advantages,along with others that will be further understood and appreciated byreference to the written disclosure, figures, and claims includedherein.

BRIEF DESCRIPTION OF THE DRAWINGS

The structure and operation of the invention will be better understoodwith the following detailed description of embodiments of the invention,along with the accompanying illustrations, in which:

FIG. 1 is a cross sectional view depicting one embodiment of a probecapable of collecting biological components;

FIG. 2 represents an elevational view of another embodiment of a probewith a guidewire lumen and side port;

FIGS. 3A and 3B are scanning electron micrographs depicting variousembodiments of the invention comprising porous structures;

FIGS. 4A through 4D are micrographs illustrating various configurationsof the micro-porous tube of a probe;

FIGS. 5A and 5B are schematic side and front elevational views of oneembodiment of the probe comprising a proximal section joined to a distalzone;

FIGS. 6A and 6B are schematic side and front elevational views ofanother embodiment of the probe comprising a unitary body design;

FIGS. 7A and 7B are schematic side and front elevational views of oneembodiment of a micro-porous probe with an atraumatic tip. FIG. 7C is alongitudinal cross sectional schematic views of one embodiment of amicro-porous probe in FIG. 7A.

FIG. 8A is a schematic of one embodiment of the probe comprising a stentwith a polymer fabric collecting surface; FIG. 8B is a cross sectionalschematic view of the probe from FIG. 8A within a blood vessel.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The detection of occult cancer cells and other biological markers hasshown promise in the diagnosis and treatment of disease. For example,the monitoring of patients' blood for circulating tumor cells and othermarkers may prove advantageous in detecting early tumor progressionbefore metastasis to other organs occurs. Circulating nucleic acids,tumor cells and proteins can be detected in the blood (inclusive ofplasma and serum), bone marrow, cavity fluids and cerebrospinal fluid(CSF) of cancer patients which may serve as risk stratification factors,markers for the presence of clinical disease, predictors of subclinicaland/or minimal residual disease presence, determinants of treatmentresponse and disease progression, and prognosticators of patientoutcome. Other body fluids shown to have the above tumor cells, proteinmarkers, carbohydrate markers or nucleic acids include urine, pleuralfluids and peritoneal fluids (ascites). However, assessment of thesemolecules/tumor cells or components thereof requires a blood sample,which is collected at a single time-point or at multiple time points bydeliberate invasion of body tissue (i.e. needle stick).

These methods are often limited by the intermittent and/or low-levelpresence of cancer cells and markers in the blood. Although newamplification and detection techniques, such as immunochemistry, flowcytometry and reverse transcriptase polymerase chain reaction, aid inthe detection of early disease markers, these techniques may fail toovercome sampling errors inherent in the blood draws. Because of theconstant circulating nature of blood and the limited volume in aparticular blood draw, evaluating a single blood sample at onetime-point may not accurately represent the quantity and quality ofcirculating nucleic acids, tumor cells, proteins or other tumor markersfor diagnosis, prognosis and monitoring of disease. Sampling error cancontribute to the frequent false-negative results found withpost-treatment cancer surveillance.

A major problem in detecting tumor cells and tumor markers in blood isthat they are not released at any particular time point. Therefore, theprobability of detecting the presence of tumor cells or markers may varyor may be unpredictable. In addition, it is known that for certainbiological markers, blood flow and release of these markers from tissuesare diurnally related and influenced by physical activity of anindividual (i.e., climbing stairs). Circulating nucleic acids, tumorcells, proteins etc as described above (here to fore termed circulatingmolecules or cells or products will be referred as markers or CMC) mayalso be released transiently into the blood stream by otherphysiological events and external influences. Repetitive samplingwithout repetitive invasive procedures would improve the accuracy andsensitivity of detecting molecules circulating in blood.

CMCs appear to circulate in varying levels/concentrations throughout aperson's disease course as well as during a single day and or inresponse to environmental manipulations such as treatment withchemotherapy, hormonal therapy, immunotherapy and radiotherapy, as wellas with administration of medications. The variations in the stabilityof these CMCs found in the blood or other body fluids add to theinherent difficulties of an assay that evaluates blood at a single timepoint. Serial assessment of blood would increase the probability ofidentifying CMCs and therefore improve their utility as prognostic,predictive and diagnostic assays. However, serial assessments ofpatients' blood require repeated patient needle sticks which areimpractical, inconvenient and uncomfortable to the patient.

A more practical and less intrusive approach would be to introduce acollecting device, probe, biomaterial adhesive matrix, chromatographyaffinity surface chip or probe, biochip, or particle into the body thatwould come in direct contact with the blood or body fluid over a periodof time. This product can then be assessed, in vivo or ex vivo, after aninterval of elapsed time to provide a more accurate evaluation of thoseCMCs. One embodiment of the invention comprises a percutaneouslyinserted device that resides indwelling in the bloodstream and is coatedwith or contains a binding partner such as nucleotides (i.e.: oligos,LNAs (locked nucleic acids), PNAs (peptide nucleic acids), cDNA, nucleicacid probes, chromatographic affinity probes or fragments thereof ortheir derivatives, complementary fragments or larger) antibodies (i.e.:monoclonal, polyclonal, FAb fragments, etc) proteins or any biologicalor synthetic material (i.e. biotin-avidin) that is complementary to theCMC in question and that can be assessed in vivo or ex vivo. The desiredbinding partner(s) are capable of binding the corresponding targetmarker of interest in a sufficient concentration and manner that permitsretrieval of the probe after an indwelling sample period of time forqualitative or quantitative analysis of the marker.

The ex vivo concept is similar to a “dip stick” approach in assessing abody fluid for a particular molecule. The in vivo concept is a like animplantable physiological monitoring device. A device and approach ofsuch nature will provide a great improvement over current methods ofevaluating blood. The evaluation of the CMC can be in the form ofconventional monitoring using established in vitro monitoring systems.For example, to detect circulating tumor cells or circulating nucleicacids, one can use RealTime quantitative PCR and oligonucleotide arrays.For detection of proteins, one can use enzyme-linked immunosorbent assay(ELISA), chromographic affinity assays, etc. For in vivo monitoring itcan be through electric or thermal related impulses or direct imaging.

The detection time of the probe may be continuous, over multipleintervals, or event-driven. Inactivating the detection mechanism attimes may conserve battery power. Reducing probe binding surfaceexposure to the body at times may also reduce fibrin deposition andother deleterious processes during periods of low yield. For example,increased core body temperature or increased serum potassium levels arecorrelated with cell lysis of certain cancers and detection during ofthese events may enhance the yield of interval collection and detectionschemes. Other event-based detection periods may include time period toassess a patient's response to therapy through detection of componentsrelated to cellular death. This allows measurement of a patient'sresponse, for example, to chemotherapy and/or radiation therapy, whichcan then be optimized to for treatment effect or to minimize sideeffects.

This device(s) can be inserted surgically, percutaneously orintravenously into the blood stream, peritoneal cavity or bone marrowsuch that continuous contact with circulating blood, and/or body fluidsis ensured. The product can then be collected for analysis in a routinefashion or monitored. Several indwelling devices are currently availablethat coexist with the patient that in long-term contact with the bloodand patients body fluids without inducing an adverse reaction. Thesedevices also do not impair everyday patient activities of daily living.These devices include but are not limited to centrally or peripherallyinserted intravenous catheters, pacemakers and their leads, automaticinternal converter defibrillators, hemodialysis catheters, peritonealcatheters and prosthetic grafts.

One example of the proposed device is a coated catheter, guidewire orfilament, chip, biomaterial and/or matrix that can be inserted through acentrally or peripherally place intravenous catheter or implantablecatheter/material into body fluids such as peritoneal cavity, bonemarrow, cerebrospinal fluid, etc. This device can then dwell incontinuous or intermittent contact with the bloodstream and/or bodyfluids to improve yield of collecting tumor cells, components thereof,circulating nucleic acids, and proteins, or other items previouslymentioned and or for prolonged or continuous in vivo or ex vivomonitoring of marker presence or activity. Monitoring time can vary invivo from one to several days to weeks or longer. This may providevaluable information on markers of subclinical and/or minimal residualcancer presence and determinants of treatment response and diseaseprogression. Such devices may also be used to monitor host states forother disease progression patterns, including but not limited toinfectious processes and organ transplant rejection.

The invention described allows for continuous invasive monitoring ofCMCs. Through a percutaneous approach, a catheter can be placed into thevasculature of a patient for continuous monitoring of circulating tumorcells and/or their component. Monitoring of CMCs may have diagnostic andprognostic value in patient care as well as serve as an improvedmechanism for monitoring response to treatment. This indwellingcatheter, for example, may be impregnated with complementary substratewhich can include but are not limited to RNA, DNA, oligonucleotides,proteins, carbohydrates, antibodies, LNAs, PNAs, probes, or anycomponent thereof and/or aforementioned in this application that hasaffinity for binding to the CMC. When the desired substrate is bound tothe catheter, chip or any device mentioned in this context containedtherein, the substrate can be quantitated and evaluated for informationthat can be conveyed to a self-embedded or external detector. Inaddition, this catheter or device (including nanoparticles, nanodevices,microfabricated devices, etc) and/or with an associated chip or otherdevice containing complementary substrate to the source(s) foridentification to which contains the bound substrate of interest can beremoved for ex vivo analysis whereby the information obtained wouldprovide both qualitative and quantitative data.

In addition to enhancing the sensitivity of detecting cancer and cancerrecurrence, the invention allows assessment of circulating tumor cellsalso would provide a rapid monitoring system to determine if a specifictherapy is effective.

In one embodiment of the invention, continuous surveillance/monitoringof circulating nucleic acids (including RNA, double stranded and singlestranded DNA, chimeric RNA/DNA), tumor cells, fetal cells, transplantallogeneic cells, transfected cells, proteins, infectious diseasenucleic acids, proteins, carbohydrates (including glucoproteins,gangliosides and phospholipids) in any complete components or fragmentforms, is performed to assess the presence and/or progression ofdisease. These molecules will be detected in serum, plasma, whole blood,bone marrow, CSF, lymphatic fluid, pleural or peritoneal fluids, urineor other body fluids in patients with cancer, hyperplasia, pregnancy(including prenatal diagnosis), patients with infectious diseasessymptomatic or asymptomatic with other medical conditions such asinfectious disease, autoimmune diseases, inflammatory diseases,cardiovascular disease (including myocardial infarction, unstable anginaand congestive heart failure), neurovascular diseases (e.g., ischemicevents, stroke, anemia), pulmonary disease (including acute respiratorydistress syndromes, fibrosis, pulmonary hypertension, emphysema, asthma,chronic obstructive pulmonary disease), renal disease (infection,hypertension nephropathies, nephritis, renal insufficiency and renalfailure), trauma patients, organ failure, critical care patients, andtransplant patients (including allogeneic and xenogeneic).

A. Binding Partners

The terms “binding partner” or “member of a binding pair” refer tomolecules that specifically bind other molecules (e.g., a marker ofinterest) to form a binding complex such as antibody-antigen,lectin-carbohydrate, nucleic acid-nucleic acid, biotin-avidin, etc. Incertain embodiments, the binding is predominantly mediated bynoncovalent (e.g. ionic, hydrophobic, etc.) interactions.

One or more binding partners that specifically bind a target marker tobe detected are affixed in the binding zone on the probe of theinvention. The binding partner(s) used in this invention are selectedbased upon the target marker(s) that are to be identified/quantified.Thus, for example, where the target marker is a nucleic acid the bindingpartner is preferably a nucleic acid or a nucleic acid binding protein.Where the target marker is a protein, the binding partner is preferablya receptor, a ligand, or an antibody that specifically binds thatprotein. Where the target marker is a sugar or glycoprotein, the bindingpartner is preferably a lectin, and so forth. A device of the inventioncan include several different types of binding partners, for example,multiple nucleic acids of different sequence and/or nucleic acidscombined with proteins in the same device. The latter would facilitate,e.g., simultaneous monitoring of gene expression at the mRNA and proteinlevels. Other combinations of different types of binding partners can beenvisioned by those of skill in the art and are within the scope of theinvention. Furthermore, the binding partner may be combined with anoptically sensitive dye to facilitate assessment of bound CMCs.

Methods of synthesizing or isolating such binding partners are wellknown to those of skill in the art. For example, nucleic acids for useas binding partners in this invention can be produced or isolatedaccording to any of a number of methods well known to those of skill inthe art. In one embodiment, the nucleic acid can be an isolatednaturally occurring nucleic acid (e.g., genomic and/or mitochondrialDNA, cDNA, mRNA, etc.). Methods of isolating naturally occurring nucleicacids are well known to those of skill in the art (see, e.g., Sambrooket al. (1989) Molecular Cloning—A Laboratory Manual (2nd Ed.), Vol. 1-3,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

1. Antibody-Based

Antibodies or antibody fragments for use as binding partners can beproduced by a number of methods well known to those of skill in the art(see, e.g., Harlow & Lane (1988) Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, and Asai (1993) Methods in Cell Biology Vol.37. Antibodies in Cell Biology, Academic Press, Inc. N.Y.). In oneembodiment, antibodies are produced by immunizing an animal (e.g., arabbit) with an immunogen containing the epitope to be detected. Anumber of immunogens may be used to produce specifically reactiveantibodies. Recombinant proteins are the preferred immunogens for theproduction of the corresponding antibodies. The antibodies may bemonoclonal or polyclonal. Naturally occurring protein may also be usedeither in pure or impure form. Synthetic peptides are also suitable andcan be made using standard peptide synthesis chemistry (see, e.g.,Barany and Merrifield, Solid-Phase Peptide Synthesis; pp. 3-284 in ThePeptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods inPeptide Synthesis, Part A., Merrifield et al. (1963) J. Am. Chem. Soc.,85: 2149-2156, and Stewart et al. (1984) Solid Phase Peptide Synthesis,2nd ed. Pierce Chem. Co., Rockford, Ill.) Preferably, human or humanizedantibodies are used to prevent host anti-xenogen antibody production.These antibodies may include antibodies derived from hybridomas (tumorcells fused with antibody-producing mammalian cells), humanizedchimerics, Epstein-Barr Virus transformed B-cells and transgenicantibodies.

Methods for producing polyclonal antibodies are also well known to thoseof skill in the art. In one embodiment, an immunogen is mixed with anadjuvant and an animal is immunized. The animal's immune response to theimmunogen preparation is monitored by taking test bleeds and determiningthe titer of reactivity to the immunogen. When sufficient titers ofantibody to the immunogen are obtained, blood is collected from theanimal and an antiserum is prepared. If desired, the antiserum can befurther fractionated to enrich for antibodies having the desiredreactivity. The animal may be a monoclonal mouse, rat, rabbit, chickenor other animal known in the art.

Monoclonal antibodies can be obtained by various techniques familiar tothose skilled in the art. In one embodiment, spleen cells from an animalimmunized with a desired antigen are immortalized, commonly by fusionwith a myeloma cell (See, Kohler and Milstein (1976) Eur. J. Immunol. 6:511-519). Alternative methods of immortalization include transformationwith Epstein Barr Virus, oncogenes, or retroviruses, or other methodswell known in the art. Colonies arising from single immortalized cellsare screened for production of antibodies of the desired specificity andaffinity for the antigen, and yields of the monoclonal antibodiesproduced by such cells can be enhanced by various techniques, includinginjection into the peritoneal cavity of a vertebrate host.Alternatively, DNA sequences encoding a monoclonal antibody or a bindingfragment thereof can be isolated by screening a DNA library from human Bcells according to the general protocol outlined by Huse et al. (1989)Science, 246: 1275-1281. Such sequences can then be expressedrecombinantly.

In one embodiment of the invention, the technique comprises attachmentof an antibody or fragment of an antibody (referred to as Ab) to adevice that can allow capture of protein, circulating tumor cells, DNAor RNA in the blood stream or body cavity. The device will be coatedwith Ab in high density. The Ab may be natural, recombinant (chimeric,Fab, scFv, etc.), or genetically engineered. Preferably the Ab will behuman to prevent anti-foreign antibody responses (i.e. human antibodyresponse to mouse antibodies; HAMA). The device can be removed afterinsertion into the blood stream to be monitored for biomarkers or cellsit can capture. The insertion device can be a catheter, array chip,capture vessel, capture filter, and/or entrapment device. The device canbe inserted for 1, 2, 3, 4 . . . 24 hrs or days or weeks. Monitoring ofthe captured biomarker or cells may be assessed in vivo or ex vivoutilizing known techniques depending on the biomarker or cell type. Thebiomarker or cells captured can be assessed quantitatively orqualitatively. In another approach the biomarker or cells captured willbe monitored in vivo utilizing a signaling indicator based onelectrical, colorimetric or activation signals.

In capturing cells the device would have specific Ab to detect cellsurface markers of cancer cells. Cancer cells have distinct markers ontheir cell surface that distinguish them from normal cells. This hasbeen demonstrated by immunohistochemistry (Racila E et al., Detectionand characterization of carcinoma cells in the blood, Proc Natl Acad SciUSA. 1998 Apr. 14; 95(8): 4589-94). These antibodies can be used totarget epithelial origin cells, tumor cells originated from specifictissues, non-epithelial origin cells (i.e. melanoma). Circulating tumorcells are found in the blood stream and body fluids of cancer patients(Hoon D S, et al., “Detection of occult melanoma cells in blood withmultiple-marker polymerase chain reaction assay” J Clin One. 1995August; 13(8); 2109-16, and Hoon D S, et al., “Molecular markers inblood as surrogate prognostic indicators of melanoma recurrence” CancerRes. 2000 Apr. 15; 60(8): 2253-7.). Tumor cells spread to distant organsvia the blood stream, lymphatic ducts or body fluids or body cavities.The spread of tumor cells can eventually lead to tumor growth at distantsites from the original tumor, thus producing metastasis. Growth ofmetatastatic tumor sites can lead to death.

Detection of tumor cells can be used as an indicator of disease spread,tumor aggressiveness, potential to spread to other organs, and presenceof disease in individuals who are otherwise diagnosed as disease-free byconventional means. Detection of tumor cells in vivo may be advantageousin some circumstances over ex vivo detection. The approach will allowbetter capture of early disease. One cannot predict disease spreading orvolume through single blood draw of a small amount of blood or bodyfluid. One approach comprises catching tumor cells through a capturingsystem placed in the blood stream or body fluid for a longer period oftime. This is may be advantageous when capturing occult circulatingmetastatic or leukemic tumor cells. The cell surface marker can be aprotein, glycoprotein, glycolipid, peptide epitope, conformationalbiological epitope or multiple disease or tumor markers. The device mayhave more than one Ab attached to it to improve sensitivity andcapturing ability. The Ab may be to multiple epitope sites of a singlebiomarker antigen. The tumor cells captured will be dislodged when thedevice is removed and assessed by the following ex vivo methods:immuno-histochemistry, DNA, mRNA and/or proteomics.

The isolation of the cells may involve physical removal or directsolvent removal specific to that biomarker's physical-chemicalproperties. For example DNA and RNA from tumor cells can be extracteddirectly from the tumor cells after isolation. Isolation of DNA or RNAcan be by solvents used for nucleic acids. This can be accomplisheddirectly or after the cells have been dislodged. RNA and DNA can bedetected by hybridization to a specific probe, polymerase chain reaction(PCR) or related monitoring approach. The assessment of nucleic acidsfrom the tumor cells can provide quantitative and qualitative analysis.Even if non tumor cells are captured, the specificity of the analysiscan be optionally increased through a second tier analysis. Sensitivityof the analysis can be further be enhanced through amplification of thenucleic acids by PCR or related methods, incorporating specific probesor detection systems ex vivo. Specificity and sensitivity ex vivo forthe specific nucleic marker can be approached using currenttechnologies. The DNA markers may include microsatellites, mutations,translocations, insertions, amplifications, SNPs or chromatin/DNAcomplexes. The RNA markers can include specific genes in whole or partin the form of mRNA.

Protein, glycoprotein, or glycolipid analysis can be detected byantibody, mass spectrophotometry, surface enhanced laserdesorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS),matrix-assisted laser desorption/ionisation-time of flight massspectrometry (MALDI-TOF MS), affinity assay, chromatographic approach.The approach can be directly from the device or removal of the biomarkerby some solvent, physical method or reagent to a vessel where it can beprocessed. The detection can be in the form of an affinity matrix chipfor the specific biomarker type.

The Ab on the device can be a natural antibody produced by human or someanimal B cells in the form of polyclonal or monoclonal antibody. The Abcan be a recombinant antibody that is released from transfectedmammalian or prokaryotic cells. The Ab can be a fragment of an antibodysuch as scFV, FV or FAb fragment that has specific recognition of thebiomarker or cell epitope. The Ab can be a genetically engineered Abthat has a specific attachment moiety or detection ability.

The Ab on the device can be polyclonal or monoclonal antibody to aspecific epitope or multiple epitopes to a specific biomarker orepitope. It can consist of multiple Ab to multiple biomarkers. Thelatter will allow higher sensitivity and capturing ability.

Ab can be attached to the device such as a catheter by direct affinityattachment, chemical attachment, biological attachment or electriccharge. The Ab can be coated in a vessel, tube or filter device, chip,filament, biopolymer matrix, biological material, capsule matrixinserted into a patient.

The Ab-coated device can be inserted into the venous, arterial orcapillary beds of a patient. It can also be inserted into a body cavitysuch as peritoneal, pleural, skin tissue, or organ/tissue cavity createdby surgical procedure.

2. Protein-Based

In one embodiment, the binding partner can be a binding protein.Suitable binding proteins include, but are not limited to, receptors(e.g., cell surface receptors), receptor ligands (e.g., cytokines,growth factors, etc.), transcription factors and other nucleic acidbinding proteins, as well as members of binding pairs, such asbiotin-avidin.

Binding proteins useful in the invention can be isolated from naturalsources, mutagenized from isolated proteins, or synthesized de novo.Means of isolating naturally occurring proteins are well known to thoseof skill in the art. Such methods include, but are not limited to,conventional protein purification methods including ammonium sulfateprecipitation, affinity chromatography, column chromatography, gelelectrophoresis and the like (see, generally, R. Scopes, (1982) ProteinPurification, Springer-Verlag, N.Y.; Deutscher (1990) Methods inEnzymology Vol. 182: Guide to Protein Purification, Academic Press, Inc.N.Y.). Where the protein binds a target reversibly, affinity columnsbearing the target can be used to affinity purify the protein.Alternatively the protein can be recombinantly expressed with a HIS-Tagand purified using Ni.sup.2+/NTA chromatography.

In another embodiment, the binding protein can be chemically synthesizedusing standard chemical peptide synthesis techniques. Where the desiredsubsequences are relatively short, the molecule may be synthesized as asingle contiguous polypeptide. Where larger molecules are desired,subsequences can be synthesized separately (in one or more units) andthen fused by condensation of the amino terminus of one molecule withthe carboxyl terminus of the other molecule thereby forming a peptidebond. This is typically accomplished using the same chemistry (e.g.,Fmoc, Tboc) used to couple single amino acids in commercial peptidesynthesizers.

The technique will involve detection of free circulating proteins,peptides or protein complexes via an affinity matrix or antibody orligand (referred to as affinity substrate; AS) coated to a device thatcan allow capture of proteins, peptides or glycoproteins in the bloodstream or body cavity. The device will be coated with AS in highdensity. The device can be removed after insertion into the blood streamto be monitored for biomarkers it can capture. The insertion device canbe a catheter, array chip, capture vessel, capture filter, entrapmentdevice. The device can be inserted for 1, 2, 3, 4 . . . 24 hrs or daysor weeks. Monitoring of the captured biomarker or cells will be assessedex vivo utilizing known techniques depending on the biomarker type. Thebiomarker captured can be assessed quantitatively or qualitatively. Inanother approach the biomarker captured will be monitored in vivoutilizing a signaling indicator based on electrical, colorimetric oractivation signals.

Protein and glycoprotein analysis can be detected ex vivo by antibody,mass spectrophotometry, affinity assay, chromatographic approach. Theapproach can be directly from the device or removal of the biomarker bysome solvent, physical method or reagent to a vessel where it can beprocessed.

The antibody used on the device can be a natural antibody produced byhuman or some animal B cells in the form of polyclonal or monoclonalantibody. The antibody can be a recombinant antibody that is releasedfrom transfected mammalian or prokaryotic cells. The antibody can be afragment of an antibody such as scFV, FV or FAb fragment that hasspecific recognition of the biomarker or cell epitope. The antibody canbe a genetically engineered antibody that has a specific attachmentmoiety or detection ability.

The AS can be in the form of affinity matrix material specific or nonspecific for specific protein properties. The former is preferable. Fornon-specific (not to a specific biomarker) the AS can be based on chargeto attract hydrophilic or hydrophobic molecules. The antibody or ligandsubstrate on the device can be towards a specific epitope or multipleepitopes to a specific biomarker. It can consist of multiple AS tomultiple biomarkers. The latter will allow higher sensitivity andcapturing ability.

AS can be attached to the device such as a catheter by methods includingbut not limited to direct affinity attachment, chemical attachment,biological attachment or electric charge. The AS can be coated in avessel, tube or filter device, chip, filament, biopolymer matrix,biological material, or capsule matrix inserted into a patient.

The AS coated device can be inserted into the venous, arterial orcapillary beds of a patient. It can be inserted into a body cavity suchas peritoneal, pleural, skin tissue, or organ/tissue cavity created bysurgical procedure.

B. Affixation of Binding Partner to Probe

The desired binding partner(s) are affixed to the binding zone on theprobe in a sufficient concentration and manner to be capable of bindingthe corresponding target marker of interest in a manner that permitsretrieval of the probe after an indwelling sample period of time andqualitative or quantitative analysis of the marker. The linkage betweenthe binding partner and the substrate surface on or attached to theprobe is preferably chemically stable under both in vivo and assayconditions. The linkage may or may not produce significant non-specificbinding of target analyte(s) to the substrate. Many methods forimmobilizing molecules to a variety of substrates are known in the art.For example, the binding partner can be covalently bound ornoncovalently attached through specific or nonspecific bonding.

If covalent bonding between a compound and the surface is desired, thesurface will usually be polyfunctional or be capable of beingpolyfunctionalized. Functional groups that may be present on thesubstrate surface and used for linking can include but are not limitedto carboxylic acids, aldehydes, amino groups, cyano groups, ethylenicgroups, hydroxyl groups, mercapto groups and the like. The manner ofcovalently linking a wide variety of compounds to various surfaces iswell known and is amply illustrated in the literature. See, for example,Ichiro Chibata (1978) Immobilized Enzymes, Halsted Press, New York, andCuatrecasas, (1970) J. Biol. Chem. 245: 3059, herein incorporated byreference.

In addition to covalent bonding, various methods for noncovalentlybonding a binding partner can be used. Noncovalent binding is typically,but not necessarily, nonspecific absorption of a compound to thesurface. Typically, the surface is blocked with a second compound toprevent nonspecific binding of labeled assay components. Alternatively,the surface is designed such that it nonspecifically binds one componentbut does not significantly bind another. For example, a surface bearinga lectin such as concanavalin A or wheat germ agglutinin will bind acarbohydrate containing compound but not an unglycosylated protein.Various substrates for use in noncovalent attachment of assay componentsare reviewed in U.S. Pat. Nos. 4,447,576 and 4,254,082, hereinincorporated by reference.

Where the binding partner is a nucleic acid or a polypeptide, themolecule can be chemically synthesized in situ, if desired. In situnucleic acid or protein synthesis typically involves standard chemicalsynthesis methods, substituting photo-labile protecting groups for theusual protecting groups (e.g., dimethoxy trityl group (DMT) used innucleic acid synthesis). Irradiation of the substrate surface atdiscrete locations results in selective coupling of the monomer (e.g.,nucleotide or amino acid) to the growing nucleic acid(s) orpolypeptide(s) at the irradiated site. Methods of light-directed polymersynthesis are well known to those of skill in the art (see, e.g., U.S.Pat. No. 5,143,854; PCT Publication Nos. WO 90/15070, WO 92/10092 and WO93/09668; and Fodor et al. (1991) Science, 251, 767-77), hereinincorporated by reference.

In one embodiment, the binding partner is immobilized to the bindingsurface by the use of a linker (e.g. a homo- or heterobifunctionallinker). Linkers suitable for joining biological binding partners areknown in the art. For example, a nucleic acid or protein molecule may belinked by any of a variety of linkers including, but not limited to apeptide linker, a straight or branched chain carbon chain linker, or bya heterocyclic carbon linker. Heterobifunctional cross linking reagentssuch as active esters of N-ethylmaleimide have been widely used (see,for example, Lemer et al. (1981) Proc. Nat. Acad. Sci. USA, 78:3403-3407 and Kitagawa et al. (1976) J. Biochem., 79: 233-236, and Birchand Lennox (1995) Chapter 4 in Monoclonal Antibodies: Principles andApplications, Wiley-Liss, N.Y.), herein incorporated by reference.

In one example, the binding partner is immobilized utilizing abiotin/avidin interaction. In this embodiment, biotin or avidin with aphotolabile protecting group can be exposed to the binding surface onthe probe. Irradiation of the surface at a distinct location results incoupling of the biotin or avidin to the surface at that location. Then,a binding partner bearing an avidin or biotin group, respectively, iscontacted with the surface, forming a biotin-avidin complex and is thuslocalized in the irradiated site. To affix multiple different bindingpartners to different locations, this process can be repeated at eachbinding partner location.

Another potential photochemical binding approach is described by Sigristet al. (1992) Bio/Technology, 10: 1026-1028, herein incorporated byreference. In this approach, the interaction of ligands with organic orinorganic surfaces is mediated by photoactivatable polymers with carbenegenerating trifluoromethyl-aryl-diazirines that serve as linkermolecules. Light activation of aryl-diazirino functions at 350 nm yieldshighly reactive carbenes, and covalent coupling is achieved bysimultaneous carbene insertion into both the ligand and the inertsurface. Thus, reactive functional groups are not required on either theligand or supporting material.

Binding partners can be affixed to any location on the surface thatcontacts the sample during an assay according to the invention. Thebinding surface on the probe may be varied considerably in form, as maybe desired based upon the binding system requirements. For example, thebinding surface may be the externally facing surface of the probe.Alternatively or in addition, as previously mentioned the probe may betubular or may comprise a porous structure to increase the surface areaavailable for the binding partner. A variety of open cell foamstructures, among others can significantly increase the effectivesurface area. Any of a variety of other surface area enhancing designtechniques may also be used, such as providing a plurality of axiallyextending fins, or a plurality of radially outwardly extendingcircumferential rings in the binding area of the probe.

C. Probe Configurations

1. Catheter-Based Probes

Referring to FIG. 1, there is disclosed a CMC or marker binding andretrieval probe 10 in accordance with one aspect of the presentinvention. Although the probe 10 will be described primarily in terms ofan insert to be temporarily placed down an existing access port orsheath into the cardiovascular system, for retrieving a marker fromblood, the present inventors contemplate broader applicability as willbe apparent to those of skill in the art in view of the disclosureherein. Existing access ports or sheaths include but are not limited toHickman catheters, Portacath catheters, peripherally inserted centralcatheter (PICC) lines, femoral, jugular, or subclavian central venouslines, radial arterial catheters and peripheral venous lines.Furthermore, additional procedures, such as transseptal puncture andtransjugular intrahepatic puncture, may be used to access other bodysites such as the arterial chambers of the heart or the portal vein,respectively.

For example, the probe may be adapted for direct access to a targetsite, without the use of a distinct tubular access catheter. In general,whether used with an access sheath or as a stand alone device, thedimensions of the probe can be optimized by persons of skill in the artin view of the present disclosure to suit any of a wide variety oftarget sites. For example, the probe of the present invention can beused to obtain samples from large and small arteries and veinsthroughout the cardiovascular system, as well as other lumens, potentialspaces, hollow organs and surgically created pathways. Marker (tumorand/or non-tumor) collection may be accomplished in blood vessels, bodylumens or cavities, such as the lymphatic system, esophagus, trachea,urethra, ureters, fallopian tubes, intestines, colon, biliary ducts,spinal canal and any other locations accessible by a flexible or rigidprobe which may contain a specific binding partner of diagnostic value.The probe 10 may also be adapted for direct advance through solidtissue, such as soft tissue or through bone, for site specificmonitoring of a binding partner of interest.

The probe 10 generally comprises an elongate body 16 extending between aproximal end 12 and a distal functional end 14. The length of the body16 depends upon the desired access site and the desired placement sitefor the distal end 14. For example, lengths in the area of from about 1cm to about 20 or 30 cm may be useful in applications that require thecatheter to be advanced down a relatively short tubular access sheath.Longer lengths may be used as desired, such as on the order of fromabout 120 cm to about 140 cm for use in percutaneous access at thefemoral artery for placement of the distal end 14 in the vicinity of thecoronary artery. Intracranial applications may call for a differentcatheter shaft length depending upon the vascular access site, as willbe apparent to those of skill in the art.

Many markers of interest, however, may be equally retrievable at anypoint throughout the cardiovascular system, in which case the probe 10may be adapted to advance down any convenient access port that may havebeen placed for other diagnostic or therapeutic use. Devices inaccordance with the present invention may also be adapted for exposureto blood by coupling to any of a variety of ports on extracorporealcirculation systems as will be apparent to those of skill in the art inview of the disclosure herein.

In the illustrated embodiment, the body 16 is divided into at least aproximal section 33 and a distal binding zone 34. In general, distalbinding zone 34 is adapted to carry a binding partner for the marker ofinterest, as will be discussed below, and may or may not be otherwisestructurally distinct from the proximal section 33.

At least the proximal section 33 of body 16 may be produced inaccordance with any of a variety of known techniques for manufacturingcatheter bodies, depending upon the desired clinical performance. Forexample, the body 16 may be formed by extrusion of any of a variety ofappropriate biocompatible polymeric materials. Known materials for thisapplication include high density polyethylene, polytetrafluoroethylene,nylons, PEEK, PEBAX and a variety of others such as those disclosed inU.S. Pat. No. 5,499,973 to Saab, the disclosure of which is incorporatedin its entirety herein by reference. Alternatively, at least a proximalportion or all of the length of body 16 may comprise a spring coil,solid walled hypodermic needle tubing, or braided reinforced wall, as isunderstood in the catheter and guidewire arts. Whether metal orpolymeric or a hybrid, the body 16 may be hollow or solid depending uponthe nature of the binding system and other desired capabilities.

In one cardiovascular example, the body 16 is provided with anapproximately circular cross-sectional configuration having an externaldiameter within the range of from about 0.025 inches to about 0.100inches. In accordance with one embodiment of the invention, the body 16has an average external diameter of about 0.042 inches (4.2 f)throughout most of its length. Alternatively, generally rectangular,oval or triangular cross-sectional configurations can also be used, aswell as other noncircular configurations, depending upon the method ofmanufacture, desired surface area, flexibility, access pathway and otherdesign considerations that may be relevant for a particular application.

Dimensions outside of the ranges identified above may also be used,provided that the functional consequences of the dimensions areacceptable for the intended purpose of the catheter. For example, thelower limit of the cross section for any portion of body 16 in a givenapplication will be a function of the number of fluid or otherfunctional lumens, if any, contained in the probe, together with thedesired surface area to be available for the binding partner, as will bediscussed.

Probe body 16 should also have sufficient structural integrity (e.g.,column strength or “pushability”) to permit the probe to be advanced toa desired target site without buckling or undesirable bending.

The proximal end 12 of the probe 10 may be provided with a grip 46 suchas a polymeric cap 48 which may be molded or otherwise secured to theproximal end 12 of the body 16. Preferably, the cap is provided with acomplementary surface structure to allow a removable connection betweenthe cap and the proximal end of the IV catheter or other device throughwhich the probe 10 will achieve contact with blood or other body fluid.Removable attachment may be accomplished by using any of a wide varietyof clips, twist fasteners such as Luer connectors, interlocking snapfitconnectors, or friction fit connectors as will be appreciated by thoseof skill in the art in view of the disclosure herein.

The axial length of the probe 10 is preferably precisely calibrated tomatch the particular access catheter with which it is to be used, toprovide a reproducible length of the binding zone to be exposed to thesample of interest.

Referring to FIG. 2, there is disclosed an alternative implementation ofthe probe of the present invention. The proximal end 12 of probe 10 isprovided with a manifold 18 having one or more access ports as is knownin the art. Manifold 18 may be provided with a guidewire port 20 in anembodiment where over-the-wire navigation of the probe may be desired.An infusion port 22 may be provided with or without the guidewire port.The infusion port is in fluid communication with the binding zonethrough an infusion lumen. This allows periodic or continuous infusionof saline, heparin or other media to prevent “clogging” or coating ofthe binding zone over time, by natural clotting or other processes whichmay interfere with the efficacy of the binding chemistry. Additionalaccess ports may be provided as needed, depending upon the desiredcapabilities of the catheter. Manifold 18 may be injection molded frommedical grade plastics or formed in accordance with other techniquesknown in the art.

The distal end 14 of the probe 10 may be provided with an atraumaticdistal tip 25 which may include a guidewire exit port 26 in a guidewirelumen embodiment as is known in the art. A radiopaque marker (notillustrated) may be provided on the probe body 16 in the case ofrelatively long probes to facilitate positioning of the probe as isknown in the art. Suitable marker bands can be produced from a varietyof materials, including platinum, gold, and tungsten/rhenium alloy.

The distal zone of the probe is provided with a binding zone, having abinding partner for binding with a marker of interest. As used herein,the term marker refers to any CMC discussed above, as well as any othercell, cell fragment, protein, peptide, glycoprotein, lipid, glycolipid,proteolipid, or other molecular or biological material that is uniquelyexpressed (e.g. as a cell surface or secreted protein) by diseasedcells, or is expressed at a statistically significant, measurablyincreased or decreased level by diseased cells, or in association with adisease state of interest (e.g. a protein expressed by an infectiousagent associated with disease), or is expressed at a statisticallysignificant, measurably increased or decreased level by diseased cellscompared to normal cells, or which is expressed by non-diseased cells inassociation with disease (e.g. in response to the presence of diseasedcells or substances produced therefrom). Disease markers can alsoinclude specific DNA or RNA sequences marking a deleterious geneticchange, conformational change compared to baseline or normal, or analteration in patterns or levels of gene expression significantlyassociated with disease. Disease markers include breast cancer markers.

The term cancer marker refers to a subset of disease markers, namely anyprotein, peptide, glycoprotein (including but not limited to mucins,mucoid and amyloid glycoproteins), lipid, glycolipid, proteolipid, orother molecular or biological material that is uniquely expressed (e.g.as a cell surface or secreted protein) by cancerous cells, or isexpressed at a statistically significant, measurably increased ordecreased level by cancerous cells compared to normal cells, or which isexpressed by non-cancerous cells in association with cancer (e.g. inresponse to the presence of cancerous cells or substances producedtherefrom). Cancer markers can also include specific DNA or RNAsequences marking a deleterious genetic change, conformational change,or an alteration in patterns or levels of gene expression significantlyassociated with cancer.

a. Binding Zone Surface Area

The binding zone may be configured with an increased surface area toprovide an increased number of binding sites on the probe. The surfacearea may be increased by providing an increased longitudinal length,increased diameter or cross-section through at least a portion of thedistal zone. In addition, or alternatively, at least a portion of thedistal zone may comprise a porous material and/or microstructure toincrease the surface area. Non-limiting examples of porous materialsinclude porous polymers, ePTFE, PTFE, polyurethane, silicone, foam, or aceramic with a porous surface (e.g., titanium nitride, titanium carbide,carbon, and silicon carbide). Various techniques for depositing materialon the probe surface to provide a porous structure may also be used andinclude ion beam deposition, sintering, sputtering, ion implantation,laser surface alloying, electroplating, physical or chemical vapordeposition, chemical or physical etching, grit blasting, plasma andthermal spray coating. Other materials that can be applied to the probesurface include iridium oxide, graphite and platinum black. The surfacearea may be increased through microstructures on the binding zonesurface, formed from processes including but not limited to mechanicalroughening of the probe surface, laser drilling or metal sintering ontothe probe. The probe may also be manufactured using microporous tubing,porous fabric and polymers, carbon fiber bundles, and nanotubes. Thesurface area of the binding zone may be configured by one skilled in theart depending upon the expected release pattern, degradation andmetabolization pathways and binding kinetics of the CMCs of interest.FIGS. 3A and 3B represent scanning electron micrographs (SEM) of variousporous configurations that provide an increased surface area for theprobe. FIG. 3A depicts one embodiment of the invention comprising amicroporous zone formed by vapor deposition. FIG. 3B depicts anotherembodiment of the invention formed with sintered metal beads. Oneskilled in the art will understand that a variety of metals may by usedfor a sintered porous surface, including but not limited to platinum,platinum/iridium and other platinum group metals or alloys thererof,titanium, titanium alloys and 316L stainless steel. In one embodiment,the sintered metal zone has an average pore size of about 5 microns toabout 150 microns to allow particle access into the microporousstructure. In other embodiments, an average pore size of about 5 micronsto about 100 microns may be used. In one example, a sintered metalporour zone has an average pore size of about 10 microns to about 50microns. Microporous structures will typically have a porosity betweenabout 10% to about 80%. In some embodiments, the porous layer has aporosity of about 10% to about 60%, and preferably about 40%. Otherbinding zone structures that increase the surface area are shown inFIGS. 4A through 4D. FIG. 4A is a photograph of a porous fabric. FIG. 4Bdepicts a porous polymer. FIG. 4C depicts laser drilled holes in apolymer surface and FIG. 4D depicts a nanotube microstructure forproviding an increased surface area.

b. Distal Tip Configurations

FIGS. 5A and 5B depict one implementation of the invention, where thebody 16 of the probe 10 comprises a proximal section 33 and a distalzone 34 attached through a joint area 50. In one embodiment, theproximal section 33 has an average outer diameter of about 0.5 mm toabout 2 mm, but average outer diameters from about 1 mm to about 30 mmmay also be used, depending on the desired location and positioningprocedure. The proximal section 33 may be made through extrusion ormolding using any of a variety of flexible biocompatible polymersincluding but not limited to PEBAX, polyurethane (Q747), PE, PTFE,nylon, silicone rubber, or combinations thereof. The polymer typicallyhave a hardness within the range of about 80A to about 75D, but polymerswithin other hardness ranges may also be used. In another embodiment,the polymer has a hardness of about 10D to about 80D. In one embodiment,the proximal section may have a length of about 20 mm to about 300 mm.In other embodiments, the proximal section may have a length of about 20cm to about 40 cm, or about 80 cm to about 140 cm, depending on thedistance from the insertion point to the target location. The joint area50 may have any of a variety of configurations for attaching theproximal section 33 and the distal zone 34, including but not limited amale/female configuration or any other mechanical or friction fit knownin the art. The proximal section 33 and distal zone 34 may be joined inany of a variety of ways, including but not limited to adhesive bondingwith medical grade epoxy, polyurethane adhesives, fast setting glue, UVcure adhesives, solvent fusing or heat fusing. A metallic core may beincluded in proximal section 33 and/or distal zone 34 to providesufficient column strength or pushability.

FIGS. 6A and 6B illustrate another embodiment of the invention where theproximal section 33 and the distal zone 34 comprise the same materialand therefore, a joint area is not required. If an increased surfacearea on the distal zone 34 is desired, laser drilling and other methodspreviously mentioned may be used to alter the surface area.

In some embodiments of the invention, an atraumatic tip is provided atthe distal end of the probe to reduce potential damage to the probe andthe surrounding tissue during insertion. FIGS. 7A through 7C representone embodiment of the probe comprising an atraumatic tip. Referring toFIG. 7A, the distal zone 34 of the probe 10 comprises a micro-poroussegment 52 joined at a joint area 50 to a soft tip 54. The soft tipcomprises a distally rounded structure comprising a material such asPEBAX, polyurethane (Q747), silicone rubber, PTFE, nylon, or otherbiocompatible polymer having a hardness within the rarige of about 80Ato about 75D. The soft or atraumatic tip 54 has a length of about 2 mmto about 6 mm and is joined at its proximal end 56 to a porous segment52 at a joint area 50 using an adhesive such as a polyurethane adhesive,an epoxy, fast setting glue, UV cure adhesive or other adhesives knownin the art. The porous segment has a length of about 1 mm to about 10 mmand an average diameter of about 0.5 mm to about 2 mm or more. Asillustrated in FIG. 7C, the porous segment 52 may comprise a ring ortubular structure, but other structures with a core may also be used.The porous ring is joined at its proximal end 58 to the proximal section33 of the probe at another joint area 50 using an adhesive or otherjoining process.

At least a portion of the porous segment 52 comprises a binding zone forinteracting with one or more CMCs. The binding zone of the distal zonemay have a diameter of about 0.5 mm to about 2 mm. In anotherembodiment, the binding zone has an average diameter of about 1 mm toabout 5 mm or more. In one embodiment, the binding zone has a length ofabout 1 mm to about 10 mm. In another embodiment, the binding zone has alength of about 5 mm to about 30 mm or more. The binding zone maycomprise a porous material and/or porous microstructure as previouslymentioned, such as a sintered metal, a porous ceramic, a sputtered orvacuum deposited metal or ceramic, a porous polymer or a laser-drilledmaterial. Further detail of the binding zone is provided below.

The body 16 of the probe 10 may optionally comprise at least one lumengenerally along the length of the body 16 for passing the probe over aguidewire. The lumen may pass from the proximal section 33 to the distalzone 34 and exit the distal end 14 of the probe 10. Alternately, thelumen may terminate prior to the distal end 14 of probe 10 at theexterior surface of the proximal section 33 or distal zone 34, similarto a rapid-exchange catheter.

2. Detachable Probes

In another embodiment of the invention, depicted in FIGS. 8A and 8B, theprobe 60 is configured so that it is capable of implantation within thebody and does not require a permanent proximal attachment formanipulation and/or retrieval of the probe 60. A detachable orimplantable probe 60 may be beneficial where detection of a CMC requiresprolonged exposure to the body, but the probe 60 is not limited to thisparticular use. By detaching from its delivery tool, contact between theprobe and the external surface of the body and the probe surface areawithin the body may be reduced. This may decrease the risk ofthrombogenicity and/or infection created by the presence of the probe60. Those with cancer or a history of cancer or other disease may bepredisposed to clot formation and infection and may benefit fromadditional measures to reduce such risks.

In one embodiment, the probe comprises a binding zone and an engagementinterface for reversibly engaging a delivery/retrieval tool. The bindingzone comprises at least one site for interacting with one or more CMC.The configuration of the binding zone is described in further detailbelow. The engagement interface comprises a mechanical or frictioninterface capable of forming a mechanical or friction fit with adelivery/retrieval tool to facilitate implantation and removal of theprobe. The engagement interface may be further configured to orient theprobe with respect the delivery/retrieval tool to facilitate positioningand removal of the probe through narrow openings such as a blood vessel.The probe may further comprise a support for maintaining theconfiguration of the binding zone and resisting deformation of thebinding zone. The support may be useful where the binding zone comprisesa thin or pliable surface. The probe may optionally comprise an anchorsystem for maintaining the position of the probe in a general orparticular location.

In one embodiment of the invention, the probe 60 comprises a stentsupport 64 attached to a binding zone jacket 62. The stent-supportcomprises 64 a first end 66, a second end 68, a lumen 70 between thefirst end and second end, and may be configured similar to a vascularstent with a mesh-like or zig-zag structure, as shown in FIGS. 8A and8B. The stent support 64 may be self-expanding or balloon-expandable.One skilled in the art will understand that any of a variety of stentstructures, configurations and materials may be used, including but notlimited to nitinol, 316L stainless steel, platinum or platinum/iridium.The stent support 64 may be dimensioned for placement in any of avariety of locations, including but not limited to cardiovascularsystem, a peripheral vein or artery, biliary system, urinary tract,gastrointestinal tract and other lumens or body cavities, natural orartificial. In one embodiment, the stent support has an average diameterof about 0.5 mm to about 2 mm. In another embodiment, the stent supporthas an average diameter of about 1 mm to about 8 mm. The stent supportmay have a length of about 5 mm to about 60 mm. In another embodiment,the stent support has a length of about 10 mm to about 30 mm.

A binding zone jacket 62 is attached to at least a portion of the stentsupport 64. The jacket 62 may surround a portion of the stent support 64or may be fixed within the lumen 70 of the stent support 64. One or morejackets may be attached to the stent 64. The jacket surface may comprisea biocompatible porous material or porous microstructure to increase thepotential binding surface area available. Biocompatible porous materialsinclude but are not limited to PEBAX, polyurethane (Q747), siliconerubber, PTFE and nylon. The configuration of the binding zone jacket isdescribed in further detail below. Alternatively, the binding zone maybe directly bonded onto the surface of the stent configuration and ajacket is not required.

Stent retrieval is known in the art and may be performed in severalways. Representative patents include but are not limited to U.S. Pat.No. 6,569,181 to Burns and U.S. Pat. No. 6,187,016 to Hedges et al.,herein incorporated in their entirety by reference. The stent supportmay further comprise one or more engagement elements to facilitateretrieval of the stent from the body by a retrieval tool.

In addition to affixing a binding partner to the binding zone, othermolecules or components may be bound to the binding zone to facilitateor support the function of the binding zone. In one embodiment, heparinis bound to the binding zone and possibly other portions of the probe toresist thrombus formation that may increasingly affect the function ofthe binding partners with extended exposure time to the body. Heparincoating of medical devices is well known in the art, as described by Hsuet al. in U.S. Pat. No. 5,417,969, herein incorporated in its entiretyby reference. In another embodiment, a streptokinase coating is providedto resist clot formation (Niku S D et al., Isolation of lymphocytes fromclotted blood, J Immunol Methods. 1987 Dec. 4; 105(1): 9-14, hereinincorporated by reference). Other materials that may be bonded to thebinding zone or probe surface include but are not limited to hydrogelsor other lubricious coatings, as described by Hostettler et al. in U.S.Pat. No. 5,919,570, and antimicrobial agents, as described by Raad andSherertz in U.S. Pat. No. 5,688,516, herein incorporated in theirentirety by reference. An antimicrobial component may reduce the risk ofprobe colonization by infectious bacterial and fungal organisms for aprobe placed into a body for an extended period of time. Suchantimicrobial agents may include but are not limited to aminoglycoside,amphotericin B, ampicillin, carbenicillin, cefazolin, cephalosporin,chloramphenicol, clindamycin, erythromycin, gentamicin, griseofulvin,kanamycin, methicillin, nafcillin, novobiocin, penicillin, polymyxin,rifampin, streptomycin, sulfamethoxazole, sulfonamide, tetracycline,trimethoprim, and vancomycin.

The probe may further comprise an optional elution zone capable ofretaining and releasing one or more substances such as drug compounds,reagents or other substances. In one embodiment, the elution zonereleases a substance that enhances release of a CMC from the body. Inanother embodiment, the elution zone releases a substance thatfacilitates detection of a CMC, including but not limited to Ab labeledfluorescent dyes. In still another embodiment, the elution zone releasesa substance capable of reducing a body's immune response to an antigenicelement on the probe. In another embodiment, the elution zone is capableof releasing one or more treatment agents for reducing fibrin depositiononto the binding zone and other portions of the probe. Fibrin depositionmay decrease or affect the binding of CMCs to their binding partnersinto the binding zone. Agents that may be released from the elution zoneinclude but are not limited to dexamethasone, paclitaxel, unfractionatedheparin, low-molecular weight heparin, enoxaprin, syntheticpolysaccharides, ticlopinin, dipyridamole, clopidogrel, fondaparinux,streptokinase, urokinase, r-urokinase, r-prourokinase, rt-PA, APSAC,TNK-rt-PA, reteplase, alteplase, monteplase, lanoplase, pamiteplase,staphylokinase, abciximab, tirofiban, orbofiban, xemilofiban,sibrafiban, roxifiban, bivalirudin, and pentoxifylline.

Alternatively, a chip-based detection system may be used. For example,DNA/oligonucleotide chip detection involves attachment or incorporationof a chip into a device to be inserted into the blood stream or bodycavity. The assessment of nucleic acids bound to the chip may beperformed in vivo directly through electronic or chemical signaling orex vivo by a detection device. For DNA analysis this will includemicrosatellite analysis for loss of heterozygosity (LOH) or by singlenucleotide polymorphism (SNP). Other genomic DNA markers can includemutations, amplifications and translocations. The analysis may involvespecific or multiple sites of the chromosomal or mitochondrial DNA fromtumor cells. RNA analysis will involve assessment of mRNA of transcriptsof specific genes related to the tumor cells. The mRNA transcript may beof the whole or part of the full transcript or a truncated derivative ofthe transcript. The procedure may also include chromatin and DNAcomplexes (histone proteins) related to specific genomic regions oftumor cells. The procedure may encompass assessing acetylation anddeacetylation of chromatin regions of specific genomic regions,methylated or non-methylated. The procedure may encompass assessingmethylated or non-methylated regions of the genomic regions such aspromoter related-regions of tumor-related genes. The chip may beinserted for 30 min, 1, 2, 3 . . . 24 hr and removed for assessment orassessed directly.

D. Insertion and Placement of Collection Probe

The collection probe may be inserted in a variety of ways and to varietyof locations within the body. In some situations, the probe may beinserted during a cancer surgery where access to sentinel sites ofdisease recurrence is readily accessible. For instance, following amastectomy and axillary node dissection for breast cancer, a collectingprobe may be implanted during the same procedure into the lymphaticducts draining the breast. Such as site may provide earlier detection ofrecurring disease and may also increase the yield from suchsurveillance. Similarly, placement of the collection probe surgicallymay also allow or subcutaneous implantation into a large vein while thepatient is still under anesthesia, thereby decreasing the risk ofinfecting the device compared to percutaneous insertion.

The device may also be configured for percutaneous insertion. Someembodiments of the device allow insertion of the probe into existinglong-term access sites such as a Hickman catheter, Portacath, or aperipherally inserted central catheter (PICC) line or variants thereof.Similarly, the probe may also be configured for insertion throughcentral venous catheters inserted into the femoral or jugular vein, orlarge-bore IV access site. For example, a Portacath is an implantablevenous access device that is frequently used in cancer patients toprovide long-term vascular access for chemotherapy. A detection probeplaced into a Portacath or a Portacath variant may serve a dual functionof treating the cancer and provide the ability to monitor treatmenteffect.

In use, a probe having at least one binding partner is provided. Theprobe is advanced to a site where a binding zone on the probe will beexposed to a carrier such as blood which may periodically contain amarker of interest. The probe is left in place for an evaluation period,to allow the marker to become bound to the binding partner. The probe isthereafter withdrawn, and evaluated to determine the presence of anymarker carried by the binding zone.

In one application, the probe is advanced through an access tube toposition the binding zone at an intralumenal site within an artery orvein. The binding zone is left at the site for an evaluation period ofgenerally at least about one hour, in come applications at least aboutfour or six hours, and for certain markers at least about 12 hours or 24hours or more. This allows collection of at least a first quantity of atarget marker from a first release of marker into the blood, and incertain applications at least also a second quantity of the targetmarker from a second release of marker into the blood, the first andsecond releases separated in time from each other. The first and secondquantities of the target marker may be collected on the same probe.Alternatively, during the evaluation period, a first probe may bewithdrawn from the site and replaced by at least a second probe, whichcarries the same or a second binding partner.

The device may be inserted through any of a variety of access methodsknown to interventional radiology, cardiology, gastroenterology andother medical and veterinary disciplines. These procedures may includebut are not limited to endoscopic retrograde cholangiopancreatography(ERCP) for placement into the biliary tree or pancreas, transseptalpuncture for placement into the arterial portion of the cardiovascularsystem, lumbar puncture into the cerebrospinal fluid, and cystoscopy forplacement into the urinary tract.

E. Ex Vivo Probe Assessment

The capture of nucleic acids from an in vivo device can be monitored exvivo using standard qualitative and quantitative molecular assays. Theassays can directly measure the nucleic acids or amplify them to measurethem. The assays can be probe-, sequence- or affinity ligand-based. Theassessment of DNA/RNA in body fluids ex vivo is known and currentlyavailable. These include but are not limited to gel electrophoresis,real time quantitative polymerase chain reaction (PCR), probe basedchromatographic assays. For DNA analysis, this will includemicrosatellite analysis for loss of heterozygosity or by singlenucleotide polymorphism (SNP). Other DNA markers can include mutations,amplifications, insertions and translocations. This may be specific ormultiple sites of the DNA from tumor cells. RNA analysis will involveassessment of mRNA of transcripts of specific genes related to the tumorcells. The mRNA transcript may be of the whole or part of the fulltranscript or a truncated derivative of the transcript. The proceduremay also include chromatin and DNA complexes related to specific genomicregions of tumor cells. The procedure may also include assessment ofacetylated and de-acetylated or modified regions of the chromatin andhistones surrounding a specific gene. The procedure may also includeassessment of methylation or demethylation of gene promoter regions.

Assessment of antibody or protein-based markers is currently availableand may include but is not limited to affinity binding assays, massspectroscopy, and ELISA. Similarly, of carbohydrate markers is alsoknown and may include affinity or ligand-bsed capture assays and massspectroscopy. One skilled in the art can select one or more assays basedupon the particular marker or markers of interest.

One embodiment of the invention comprises a percutaneously insertabledevice affixed with antibodies that recognize tumor-related cell surfaceproteins/glycoproteins (i.e.: cMet, HER2/neu, beta-Human chorionicgonadotropin (HCG), MUC-1, etc) or glycolipids (gangliosides GM2, GD2).The antibodies can capture and bind the circulating tumor cells in theblood or body fluid. Single or multiple antibodies to a specific cellsurface marker or multiple markers may be used. The capture device orcatheter with the bound tumor cells can be removed and subjected tostandard ex vivo isolation methods known in the art for RNA, DNA,carbohydrate and protein isolation and purification. The isolation ofthese cell products is one approach to identify their specificity.Another approach is to isolate the cells and assess them as whole cells.These approaches are advantageous in providing a unique in vivoenrichment method for the collection of circulating tumor cells andtheir subcomponents, such as DNA, RNA and proteins, for furtherevaluation and assessment.

In some embodiments, the cells can be removed physically, biochemicallyor eluted off the device by competitive reagents to the antibody.Preferably, once eluted, the cells can undergo respective componentisolation. In other embodiments, the cells are analyzed while stillattached to the device. In one example, cells can be processed, purifiedand quantitated for specific nucleic acids such as RNA and DNA bymethods known in the art. To assess the amount of nucleic acids, one canperform qualitative and/or quantitative analysis for specific RNA andDNA markers that are tumor-related. These markers may be different fromthe antibody specific markers that are used to capture the cells. Theantibody used to capture markers may also be used.

In one embodiment, cell capture with antibody to c-Met is performed andthen assessment for cMet mRNA expression is performed qualitatively orquantitatively by realtime PCR. PCR provides amplification of the targetmRNA marker and allows for detection through many available approachesincluding but not limited to as gel electrophoresis, realtime PCRthermocyclers, etc.

In one embodiment, tumor mRNA markers for assessment can include markersmost prevalent in the type of cancer being assessed, but less prevalentmarkers may also be used. For example, in melanoma one could assess forMART-1 mRNA. For breast cancer one can assess mammoglobin. Quantitativemarker detection may be used to rule out false positives. This providesanother layer of specificity to the detection scheme. Also, to increasethe sensitivity of the detection scheme, multiple markers can be used toassess for isolated tumor cells. One can also assess for specific DNAmarkers such as mutations, loss of heterozygosity, amplification,translocation, etc. Specific genetic changes may be related to specificcancers or groups of cancers. Specific genetic changes can be used incombination with multiple marker detection approaches. Some examplesinclude detection of BRAF mutation at V600 for melanoma, methylation ofRASSF1a promoter site, or LOH at 9p21. The use of specific nucleicmarkers can be used to determine specific types of cancers, level ofdisease malignancy, disease aggressiveness, prognostic and predictivevalues and other information.

In one approach, proteins are isolated and purified by direct isolation.These proteins can be assessed by ELISA for specific tumor markers,Western Blot approaches, mass spectrometry, protein arrays,ProteinChips, antibody based assays, affinity protein based assays, etcin a quantitative and qualitative manner. The approaches can be used forglycoproteins and other carbohydrate markers. The use of specificprotein/glycoprotein/carbohydrate markers can be used to determinespecific types of cancers, level of disease malignancy, diseaseaggressiveness, prognostic and predictive values and other information.

Another approach is to elute the cells. Cells bound to the catheter canbe evaluated using conventional histopathologic and immunocytochemicalstaining methods that characterize the collected cells of interest.These cells can be evaluated directly on the catheter or, in oneembodiment, the cells are isolated from the catheter using standardmethods to disrupt tumor cell complementary antibody binding throughcurrent methods of mechanical separation (such as scraping and/orwashings with saline, buffered solutions, or media). In anotherembodiment, chemical dissociation techniques are used and includewashing the catheter/antibody/tumor cell complex with pH bufferedsolutions (such as PBS with EDTA or salts that disrupt antibody bindingto cells but not destroy the cells, etc.), thus allowing the cells to becollected intact after separation from the catheter/antibody complex andassessed by conventional methods such as immunostaining procedures. Instill another embodiment, cells may also be released by disrupting theantibody-cell complex from the device. After isolation, the cells can beimmunostained with specific antibodies against tumor cell surfacemarkers or intracellular markers. The assessment of tumor cells may beperformed by conventional immunopathology for tumor cell diagnosis, butother approaches are known in the art, including but no limited toimmunostained cells by FACs analysis. In these approaches, multipleantibodies can be used for detection to improve sensitivity andspecificity for specific cells. Also, some approaches allow detection ofthe number of cells detected for quantitation of disease level. Cellscan be also assessed by conventional or non-conventional stains and dyesthat are not antibody-based. Still another approach is in situhybridization with nucleic acids or derivative molecules that arecomplimentary. The above approaches for detection of eluted cells,intact or not intact, for specific components (protein, nucleic acids,etc) can be approached quantitatively or qualitatively. The approachescan be by individual or combination of methods.

While this invention has been particularly shown and described withreferences to embodiments thereof, it will be understood by thoseskilled in the art that various changes in form and details may be madetherein without departing from the scope of the invention. For all ofthe embodiments described above, the steps of the methods need not beperformed sequentially.

1. A biological surveillance probe for detecting disease, comprising anelongate body having a proximal end and a distal end; a binding surfaceattached to the elongate body, wherein the binding surface has amicroconfiguration for an increased surface area; and at least onebinding partner attached to the binding surface to bind at least onecomplementary target.
 2. The probe of claim 1, wherein the bindingsurface is a microporous surface.
 3. The probe of claim 1, wherein thebinding surface has at least one laser-drilled hole.
 4. The probe ofclaim 1, wherein the binding surface has been configured by vapordeposition.
 5. The probe of claim 1, wherein the binding surface hasbeen configured by physical vapor deposition.
 6. The probe of claim 19,wherein the binding surface has been configured by chemical vapordeposition.
 7. The probe of claim 1, wherein the binding surface hasbeen configured by sputtering.
 8. The probe of claim 7, wherein thebinding surface has been configured by reactive sputtering.
 9. The probeof claim 1, wherein the binding surface has been configured bysintering.
 10. The probe of claim 1, wherein the binding surface hasbeen configured by vacuum deposition.
 11. The probe of claim 2, whereinthe binding surface comprises a material selected from a groupcomprising a microporous polymer, nanotube, metal, non-metal, ceramic orcombination thereof.
 12. The probe of claim 1, wherein the elongate bodyis a catheter body.
 13. The probe of claim 1, wherein the elongate bodyis a stent support.
 14. The probe of claim 13, wherein the bindingsurface is polymeric jacket.
 15. The probe of claim 1, furthercomprising at least one optically sensitive dye engaged to the bindingsurface.
 16. The probe of claim 1, further comprising afibrin-deposition resistant component.
 17. The probe of claim 1, furthercomprising at least one anti-thrombotic agent engaged to the bindingsurface.
 18. The probe of claim 1, further comprising at least oneantimicrobial agent engaged to the binding surface.
 19. The probe ofclaim 1, further comprising an atraumatic tip attached to the distal endof the elongate body.
 20. A method for collecting biological markers,comprising the steps of: providing a collecting probe comprising amicroconfigured binding surface and at least one binding agent affixedto the binding surface for binding a marker; positioning at least aportion of the probe in an anatomical structure of a living organism;maintaining the probe in a general position for a specified period oftime; and removing the probe from the living organism.
 21. The method ofclaim 20, further comprising: binding at least one marker at a firstpoint in time; and binding at least one marker at a second point intime.
 22. The method of claim 20, further comprising: binding at leastone marker at about a first peak in marker concentration; and binding atleast the biological marker at about a second peak in markerconcentration.
 23. The method of claim 20, further comprising analyzingthe probe for markers bound to the binding agent.
 24. The method ofclaim 23, wherein the analyzing step is performed ex vivo.